JP6849720B2 - Glucagon analogue - Google Patents

Description

The present invention relates to glucagon analogs and their medical use, for example, in the treatment of obesity, overweight, diabetes, and other metabolic disorders.

Preproglucagon is a precursor polypeptide of 158 amino acids, which undergoes various treatments in tissues to be glucagon (Glu), glucagon-like peptide-1 (GLP-1), and glucagon-like peptide-2 (GLP-2). ), And many structurally related proglucagon-derived peptides, including oxintomodulin (OXM). These molecules are involved in a wide variety of physiological functions, including glucose homeostasis, insulin secretion, gastric emptying, and intestinal growth, as well as regulation of food intake.

Glucagon is a 29 amino acid peptide corresponding to amino acid positions 53-81 of preproglucagon. Oxintmodulin (OXM) contains 37 octapeptides, including carboxy-terminal extension (positions 82-89 of preproglucagon amino acids called "intervening peptide 1" or IP-1) and the complete 29 amino acid sequence of glucagon. Amino acid peptide. The major bioactive fragment of GLP-1 is produced as a C-terminal amidated peptide of 30 amino acids corresponding to amino acids 98-127 of preproglucagon.

Glucagon helps maintain blood glucose levels through glycogenolysis, which binds to glucagon receptors on hepatocytes and releases glucose stored in the form of glycogen from the liver. When these stores are depleted, glucagon stimulates the liver to synthesize additional glucose by gluconeogenesis. This glucose is released into the bloodstream and prevents the development of hypoglycemia.

GLP-1 reduces elevated blood glucose levels by increasing glucose-stimulated insulin secretion and promotes weight loss primarily through reduced food intake.

OXM is released into the blood in response to the digestion of food and in proportion to the amount of calories in the diet. OXM has been shown to suppress human appetite and inhibit food intake (Cohen et al, Journal of Endocrinology and Metabolism, 88, 4696-4701, 2003; WO 2003/022304). Rats treated with oxintomodulin gain less weight than control rats, so in addition to an appetite-suppressing effect similar to that of GLP-1, OXM also affects body weight by another mechanism. It seems (Bloom, Endocrinology 2004, 145, 2687). In addition, treatment of obese rodents with OXM improves their glucose tolerance (Parlevliet et al, Am J Physiol Endocrinol Metab, 294, E142-7, 2008) and suppresses weight gain (International Publication No. 2003/022304). Pamphlet).

OXM activates both glucagon and GLP-1 receptors (2x more potent than Glucagon receptors), but naturally present on each of these receptors. And less potent than natural GLP-1. Human glucagon can also activate both receptors and show a stronger priority over glucagon receptors than GLP-1 receptors. On the other hand, GLP-1 cannot activate the glucagon receptor. The mechanism of action of oxintomodulin is not well understood. In particular, it is unclear whether the extrahepatic effects of this hormone are partially mediated by GLP-1 or glucagon receptors, or through one or more unidentified receptors.

Other peptides bind to and activate both glucagon and GLP-1 receptors (Hjort et al, Journal of Biological Chemistry, 269, 30121-30124, 1994), suppress weight gain, and ingest food. Has been shown to reduce (eg, WO 2006/134340, 2007/100535, 2008/10101, 2008/152403, 2009/155257, 2009/155258, 2010/070252, 2010/070253, 2010/070255, 2010/070251, 2011/006497, 2011/160630, 2011/160633, 2013/092703, 2014 See the 041195 pamphlet).

Obesity is a growing global health problem associated with a variety of diseases, especially cardiovascular disorders (CVD), type II diabetes, obstructive sleep apnea, certain cancers, and osteoarthritis. As a result, obesity has been shown to shorten lifespan. According to a 2005 forecast by the World Health Organization, 400 million adults worldwide (those over the age of 15) are classified as obese. In the United States, obesity is considered to be the second leading cause of preventable death after smoking.

Increased obesity leads to increased diabetes, and about 90% of people with type II diabetes can be classified as obese. There are 246 million diabetics worldwide, and it is estimated that 380 million will be diabetic by 2025. Many also have other cardiovascular risk factors, including high / abnormal LDL, high / abnormal triglyceride and low HDL.

In the first aspect, the present invention relates to a compound having the ^{formula R 1} -XZR ^{2 [in the formula}
R ¹ is H (ie hydrogen), C _1-4 alkyl, acetyl, formyl, benzoyl, or trifluoroacetyl;
R ² is OH or NH ₂ ;
X is an array:
It is a peptide having H-X2-X3-GTFTSDYSKYLD-X16-X17-AA-X20-DFV-X24-WLL-X28-A;
X2 is selected from Ala, D-Ala, Ser, N-Me-Ser, Ac3c, Ac4c, and Ac5c;
X3 is selected from Gln and His;
X16 is selected from Ser and Ψ;
X17 is selected from Lys and Ψ;
X20 is selected from His and Ψ;
X24 is selected from Glu and Ψ;
X28 is selected from Ser and Ψ;
When X2 is Ser, X3 is His;
Each Ψ is a residue independently selected from Lys, Arg, Orn and Cys, and the side chain of each residue Ψ is attached to a lipophilic substituent;
Z is a sequence of 1 to 20 amino acid units that does not exist or is independently selected from the group consisting of Ala, Leu, Ser, Thr, Tyr, Cys, Glu, Lys, Arg, Dbu, Dpr, and Orn. Is]
Alternatively, a pharmaceutically acceptable salt or solvate thereof is provided.

In some embodiments, X17 is Ψ.

In some embodiments, only X17 is Ψ, i.e. this compound comprises one or only residue Ψ present at position 17.

Peptide X
HAQGTFTSDYSKYLDSKAAHDFVEWLLSA
H-NMeSer-QGTFTSDYSKYLDSKAAHDFVEWLLSA
H-Ac3c-QGTFTSDYSKYLDSKAAHDFVEWLLSA
H-Ac4c-QGTFTSDYSKYLDSKAAHDFVEWLLSA
HSHGTFTSDYSKYLDSKAAHDFVEWLLSA
HAHGTFTSDYSKYLDSKAAHDFVEWLLSA
H-DAla-HGTFTSDYSKYLDSKAAHDFVEWLLSA
H-Ac3c-HGTFTSDYSKYLDSKAAHDFVEWLLSA and
H-Ac4c-HGTFTSDYSKYLDSKAAHDFVEWLLSA
Or
HAQGTFTSDYSKYLDSΨAAHDFVEWLLSA
H-NMeSer-QGTFTSDYSKYLDSΨAAHDFVEWLLSA
H-Ac3c-QGTFTSDYSKYLDSΨAAHDFVEWLLSA
H-Ac4c-QGTFTSDYSKYLDSΨAAHDFVEWLLSA
HSHGTFTSDYSKYLDSΨAAHDFVEWLLSA
HAHGTFTSDYSKYLDSΨAAHDFVEWLLSA
H-DAla-HGTFTSDYSKYLDSΨAAHDFVEWLLSA
H-Ac3c-HGTFTSDYSKYLDSΨAAHDFVEWLLSA and
H-Ac4c-HGTFTSDYSKYLDSΨAAHDFVEWLLSA
It may have a sequence selected from.

The compound of the present invention
H-HAQGTFTSDYSKYLDSKAAHDFVEWLLSA-NH ₂
HH-NMeSer-QGTFTSDYSKYLDSKAAHDFVEWLLSA-NH ₂
HH-Ac3c-QGTFTSDYSKYLDSKAAHDFVEWLLSA-NH ₂
HH-Ac4c-QGTFTSDYSKYLDSKAAHDFVEWLLSA-NH ₂
H-HSHGTFTSDYSKYLDSKAAHDFVEWLLSA-NH ₂
H-HAHGTFTSDYSKYLDSKAAHDFVEWLLSA-NH ₂
HH-DAla-HGTFTSDYSKYLDSKAAHDFVEWLLSA-NH ₂
HH-Ac3c-HGTFTSDYSKYLDSKAAHDFVEWLLSA-NH ₂ and
HH-Ac4c-HGTFTSDYSKYLDSKAAHDFVEWLLSA-NH ₂
Or
H-HAQGTFTSDYSKYLDSΨAAHDFVEWLLSA-NH ₂
HH-NMeSer-QGTFTSDYSKYLDSΨAAHDFVEWLLSA-NH ₂
HH-Ac3c-QGTFTSDYSKYLDSΨAAHDFVEWLLSA-NH ₂
HH-Ac4c-QGTFTSDYSKYLDSΨAAHDFVEWLLSA-NH ₂
H-HSHGTFTSDYSKYLDSΨAAHDFVEWLLSA-NH ₂
H-HAHGTFTSDYSKYLDSΨAAHDFVEWLLSA-NH ₂
HH-DAla-HGTFTSDYSKYLDSΨAAHDFVEWLLSA-NH ₂
HH-Ac3c-HGTFTSDYSKYLDSΨAAHDFVEWLLSA-NH ₂ and
HH-Ac4c-HGTFTSDYSKYLDSΨAAHDFVEWLLSA-NH ₂
May be selected from.

Aspect 2
In a second aspect, the present invention relates to a compound having the ^{formula R 1} -XZR ^{2 [in the formula,}
R ¹ is H (ie hydrogen), C _1-4 alkyl, acetyl, formyl, benzoyl, or trifluoroacetyl;
R ² is OH or NH ₂ ;
X is an array:
It is a peptide having X1-X2-X3-GTFTSDYSKYL-X15-X16-X17-X18-A-X20-DFI-X24-WLE-X28-X29;
X1 is selected from His and Tyr;
X2 is selected from Aib, D-Ser, Ala, D-Ala, Abu, Pro, Ac3c, Ac4c, and Ac5c;
X3 is selected from Gln and His;
X15 is selected from Asp and Glu;
X16 is selected from Glu, Lys and Ψ;
X17 is selected from Lys, Arg and Ψ;
X18 is selected from Ala and Arg;
X20 is selected from Lys, His and Ψ;
X24 is selected from Glu, Lys and Ψ;
X28 is selected from Ser, Glu, Lys and Ψ;
X29 is selected from Ala and Glu;
Each Ψ is a residue independently selected from Lys, Arg, Orn and Cys, and the side chain of each residue Ψ is attached to a lipophilic substituent;
Z is a sequence of 1 to 20 amino acid units that does not exist or is independently selected from the group consisting of Ala, Leu, Ser, Thr, Tyr, Cys, Glu, Lys, Arg, Dbu, Dpr, and Orn. Is]
Alternatively, a pharmaceutically acceptable salt or solvate thereof is provided.

In some embodiments of the second aspect, Ψ is present in any of X16, X17, X20, X24 and X28. In some cases, Ψ is present in at least one of X16, X17, X20, X24 and X28. It is desirable that this is the only residue Ψ present in the molecule.

Aspect 2.1
In some embodiments of the second aspect,
X1 is His;
X2 is selected from D-Ser, Ala, D-Ala, Abu, Pro, Ac3c, Ac4c, and Ac5c;
X3 is selected from Gln and His;
X16 is selected from Glu, Lys and Ψ;
X17 is selected from Lys, Arg and Ψ;
X18 is selected from Ala and Arg;
X20 is selected from Lys and Ψ;
X24 is selected from Glu, Lys and Ψ;
X28 is selected from Ser, Lys and Ψ;
X29 is Ala.

In some embodiments of the second aspect, Ψ is present in any of X16, X17, X24 and X28. In some cases, Ψ is present in at least one of X16, X17, X24 and X28. It may be desirable for this to be the only residue Ψ present in the molecule.

Aspect 2.1.1
In a particular example
X1 is His;
X2 is selected from Ala, D-Ala, Abu and Pro;
X3 is Gln;
X16 is selected from Glu and Ψ;
X17 is selected from Lys and Ψ;
X18 is Ala;
X20 is selected from Lys and Ψ;
X24 is selected from Glu and Ψ;
X28 is selected from Ser and Ψ;
X29 is Ala.

Peptide X
H-Abu-QGTFTSDYSKYLDEKAAKDFIEWLESA
HAQGTFTSDYSKYLDEKAAKDFIEWLESA
H-DAla-QGTFTSDYSKYLDEKAAKDFIEWLESA and
HPQGTFTSDYSKYLDEKAAKDFIEWLESA
Or
H-Abu-QGTFTSDYSKYLDEΨAAKDFIEWLESA
HAQGTFTSDYSKYLDEΨAAKDFIEWLESA
H-DAla-QGTFTSDYSKYLDEΨAAKDFIEWLESA and
HPQGTFTSDYSKYLDEΨAAKDFIEWLESA
It may have a sequence selected from.

The compound of the present invention
HH-Abu-QGTFTSDYSKYLDEKAAKDFIEWLESA-NH ₂
H-HAQGTFTSDYSKYLDEKAAKDFIEWLESA-NH ₂
HH-DAla-QGTFTSDYSKYLDEKAAKDFIEWLESA-NH ₂ and
H-HPQGTFTSDYSKYLDEKAAKDFIEWLESA-NH ₂
Or
HH-Abu-QGTFTSDYSKYLDEΨAAKDFIEWLESA-NH ₂
H-HAQGTFTSDYSKYLDEΨAAKDFIEWLESA-NH ₂
HH-DAla-QGTFTSDYSKYLDEΨAAKDFIEWLESA-NH ₂ and
H-HPQGTFTSDYSKYLDEΨAAKDFIEWLESA-NH ₂
May be selected from.

Aspect 2.1.2
In an alternative example
X1 is His;
X2 is selected from Ac3c, Ac4c, and Ac5c;
X3 is selected from Gln and His;
X16 is selected from Glu, Lys and Ψ;
X17 is selected from Lys, Arg and Ψ;
X18 is selected from Ala and Arg;
X20 is selected from Lys and Ψ;
X24 is selected from Glu, Lys and Ψ;
X28 is selected from Ser, Lys and Ψ;
X29 is Ala.

Peptide X
H-Ac4c-QGTFTSDYSKYLDEKAAKDFIEWLESA
H-Ac4c-QGTFTSDYSKYLDEKRAKDFIEWLESA
H-Ac4c-QGTFTSDYSKYLDKRAAKDFIEWLESA
H-Ac4c-QGTFTSDYSKYLDEKRAKDFIEWLESA
H-Ac4c-QGTFTSDYSKYLDERAAKDFIKWLESA
H-Ac4c-QGTFTSDYSKYLDERRAKDFIKWLESA
H-Ac4c-QGTFTSDYSKYLDERAAKDFIEWLEKA
H-Ac4c-QGTFTSDYSKYLDERRAKDFIEWLEKA
H-Ac4c-HGTFTSDYSKYLDEKAAKDFIEWLESA and
H-Ac4c-HGTFTSDYSKYLDEKRAKDFIEWLESA
Or
H-Ac4c-QGTFTSDYSKYLDEΨAAKDFIEWLESA
H-Ac4c-QGTFTSDYSKYLDEΨRAKDFIEWLESA
H-Ac4c-QGTFTSDYSKYLDΨRAAKDFIEWLESA
H-Ac4c-QGTFTSDYSKYLDEΨRAKDFIEWLESA
H-Ac4c-QGTFTSDYSKYLDERAAKDFIΨWLESA
H-Ac4c-QGTFTSDYSKYLDERRAKDFIΨWLESA
H-Ac4c-QGTFTSDYSKYLDERAAKDFIEWLEΨA
H-Ac4c-QGTFTSDYSKYLDERRAKDFIEWLEΨA
H-Ac4c-HGTFTSDYSKYLDEΨAAKDFIEWLESA and
H-Ac4c-HGTFTSDYSKYLDEΨRAKDFIEWLESA
It may have a sequence selected from.

The compound of the present invention
HH-Ac4c-QGTFTSDYSKYLDEKAAKDFIEWLESA-NH ₂
HH-Ac4c-QGTFTSDYSKYLDEKRAKDFIEWLESA-NH ₂
HH-Ac4c-QGTFTSDYSKYLDKRAAKDFIEWLESA-NH ₂
HH-Ac4c-QGTFTSDYSKYLDEKRAKDFIEWLESA-NH ₂
HH-Ac4c-QGTFTSDYSKYLDERAAKDFIKWLESA-NH ₂
HH-Ac4c-QGTFTSDYSKYLDERRAKDFIKWLESA-NH ₂
HH-Ac4c-QGTFTSDYSKYLDERAAKDFIEWLEKA-NH ₂
HH-Ac4c-QGTFTSDYSKYLDERRAKDFIEWLEKA-NH ₂
HH-Ac4c-HGTFTSDYSKYLDEKAAKDFIEWLESA-NH ₂ and
HH-Ac4c-HGTFTSDYSKYLDEKRAKDFIEWLESA-NH ₂
Or
HH-Ac4c-QGTFTSDYSKYLDEΨAAKDFIEWLESA-NH ₂
HH-Ac4c-QGTFTSDYSKYLDEΨRAKDFIEWLESA-NH ₂
HH-Ac4c-QGTFTSDYSKYLDΨRAAKDFIEWLESA-NH ₂
HH-Ac4c-QGTFTSDYSKYLDEΨRAKDFIEWLESA-NH ₂
HH-Ac4c-QGTFTSDYSKYLDERAAKDFIΨWLESA-NH ₂
HH-Ac4c-QGTFTSDYSKYLDERRAKDFIΨWLESA-NH ₂
HH-Ac4c-QGTFTSDYSKYLDERAAKDFIEWLEΨA-NH ₂
HH-Ac4c-QGTFTSDYSKYLDERRAKDFIEWLEΨA-NH ₂
HH-Ac4c-HGTFTSDYSKYLDEΨAAKDFIEWLESA-NH ₂ and
HH-Ac4c-HGTFTSDYSKYLDEΨRAKDFIEWLESA-NH ₂
May be selected from.

Aspect 2.2
In an alternative embodiment of the second aspect,
X1 is His;
X2 is Aib;
X3 is Gln;
X15 is selected from Asp and Glu;
X16 is selected from Glu, Lys and Ψ;
X17 is Arg;
X18 is Ala;
X20 is selected from Lys, His and Ψ;
X24 is selected from Glu, Lys and Ψ;
X28 is selected from Ser and Ψ;
X29 is Ala.

In some embodiments, one of X16 and X24 is Lys or Ψ and the other is Glu.

Additional or alternative, X15 is Glu and X16 is Lys or Ψ.

Peptide X
H-Aib-QGTFTSDYSKYLDKRAAKDFIEWLESA
H-Aib-QGTFTSDYSKYLDERAAKDFIKWLESA
H-Aib-QGTFTSDYSKYLEKRAAKDFIEWLESA and
H-Aib-QGTFTSDYSKYLEKRAAHDFIEWLESA
Or
H-Aib-QGTFTSDYSKYLDΨRAAKDFIEWLESA
H-Aib-QGTFTSDYSKYLDERAAKDFIΨWLESA
H-Aib-QGTFTSDY SKYLE ΨRAAK DIEWLESA and
H-Aib-QGTFTSDYSKYLEΨRAAHDFIEWLESA
It may have a sequence selected from.

The compound of the present invention
HH-Aib-QGTFTSDYSKYLDKRAAKDFIEWLESA-NH ₂
HH-Aib-QGTFTSDYSKYLDERAAKDFIKWLESA-NH ₂
HH-Aib-QGTFTSDYSKYLEKRAAKDFIEWLESA-NH ₂ and
HH-Aib-QGTFTSDYSKYLEKRAAHDFIEWLESA-NH ₂
Or
HH-Aib-QGTFTSDYSKYLDΨRAAKDFIEWLESA-NH ₂
HH-Aib-QGTFTSDYSKYLDERAAKDFIΨWLESA-NH ₂
HH-Aib-QGTFTSDYSKYLEΨRAAKDFIEWLESA-NH ₂ and
HH-Aib-QGTFTSDYSKYLEΨRAAHDFIEWLESA-NH ₂
May be selected from.

Aspect 2.3
In an alternative embodiment of the second aspect,
X1 is Tyr;
X2 is Aib;
X3 is Gln;
X16 is selected from Glu and Ψ;
X17 is selected from Lys and Ψ;
X18 is Ala;
X20 is selected from Lys and Ψ;
X24 is selected from Glu and Ψ;
X28 is selected from Ser and Ψ;
X29 is Ala.

Peptide X has the sequence:
Y-Aib-QGTFTSDYSKYLDEKAAKDFIEWLESA
Or
Y-Aib-QGTFTSDYSKYLDEΨAAKDFIEWLESA
May have.

The compound of the present invention
HY-Aib-QGTFTSDYSKYLDEKAAKDFIEWLESA-NH ₂
Or
HY-Aib-QGTFTSDYSKYLDEΨAAKDFIEWLESA-NH ₂
It may be.

Aspect 2.4
In an alternative embodiment of the second aspect, both X28 and X29 are Glu.

For example
X1 is His;
X2 is Aib;
X3 is Gln;
X16 is selected from Glu and Ψ;
X17 is selected from Lys and Ψ;
X18 is selected from Ala and Ψ;
X20 is selected from Lys and Ψ;
X24 is selected from Glu and Ψ;
X28 is Glu;
The X29 is a Glu.

Peptide X has the sequence:
H-Aib-QGTFTSDYSKYLDEKAAKDFIEWLEEE
Or
H-Aib-QGTFTSDYSKYLDEΨAAKDFIEWLEEE
May have.

The compounds of the present invention are:
HH-Aib-QGTFTSDYSKYLDEKAAKDFIEWLEEE-NH ₂
Or
HH-Aib-QGTFTSDYSKYLDEΨAAKDFIEWLEEE-NH ₂
It may be.

To avoid doubt, in all aspects of the invention, positions not explicitly stated to be different are fixed and may therefore contain only the residues described.

In all embodiments, the compounds of the invention may comprise one or more residues Ψ. As described in more detail below, each residue Ψ is a residue independently selected from Lys, Arg, Orn and Cys, and the side chain of each residue Ψ is attached to a lipophilic substituent. are doing.

It may be desirable for the compounds of the invention to contain no more than three residues Ψ or no more than two residues Ψ. In particular, it may be desirable for the compounds of the invention to contain one or less residues Ψ, i.e., no residues Ψ or only one residue Ψ.

The lipophilic substituent is typically attached to a functional group at the end of the side chain distal to the alpha carbon. The ability of the side chains involved in the functional group-mediated interactions (eg, intramolecular and intermolecular interactions) can be diminished or completely eliminated by the presence of lipophilic substituents. Therefore, the overall properties of the compound may be relatively unaffected by the differences in the actual amino acids present as residual Ψ. Therefore, it is considered that arbitrary residues Lys, Arg, Orn, and Cys can exist at any position where Ψ can exist. However, in certain embodiments, it may be advantageous for the amino acid component of Ψ to be Lys.

If the residue Ψ is present, the side chain of the residue Lys, Arg, Orn or Cys is attached to a lipophilic substituent.

The lipophilic substituent is the formula Z ¹ (where Z ¹ is the lipophilic moiety directly attached (covalently) to the side chain of the Lys, Arg, Orn or Cys residue), or Z ¹ Z. ^{It can have 2} (in the formula, Z ¹ is the lipophilic moiety, Z ² is the spacer, and Z ¹ is attached to the side chain of the residue via ^{Z 2).}

In any aspect of the invention, R ¹ can be selected from H, and C _1-4 alkyl (eg, methyl).

For peptide sequences X or X-Z consisting only of naturally occurring amino acids, the present invention further provides nucleic acids (which may be DNA or RNA) encoding peptide X or X-Z as defined herein. For compounds containing a residue Ψ consisting of a lipophilic moiety attached to a Lys, Arg or Cys residue, the nucleic acid may encode the appropriate Lys, Arg or Cys at the relevant position.

Also provided are an expression vector containing such a nucleic acid and a host cell containing such a nucleic acid or expression vector. The host cell is typically capable of expressing and optionally secreting the encoded peptide X or X-Z.

The compounds of the present invention are glucagon analog peptides. As used herein, the term glucagon analog peptide should be understood to refer to the compounds of the invention or peptides X or XZ as defined in the context of this specification. When referred to as a compound in the present invention, any pharmaceutically acceptable salt (eg, acetate or chloride salt) or solvent of the compound of the present invention is used unless otherwise specified or excluded from the context. It should be understood that it includes Japanese products.

The present invention includes compounds of the invention as defined herein, including pharmaceutically acceptable salts or solvates thereof as previously described, nucleic acids encoding peptide X or XZ, such nucleic acids. Provided are a composition comprising an expression vector containing, or a host cell containing such a nucleic acid or expression vector in a mixture with a carrier. In a preferred embodiment, the composition is a pharmaceutical composition and the carrier is a pharmaceutically acceptable carrier. The glucagon analog peptide may also be in the form of a pharmaceutically acceptable salt of the glucagon analog.

The compounds described herein can be specifically utilized to prevent weight gain or promote weight loss. "Preventing" means suppressing or reducing weight gain compared to the absence of treatment, and does not necessarily mean a complete cessation of weight gain. The peptide can cause reduced food intake and / or increased energy expenditure, resulting in observed effects on body weight. Independent of their effects on body weight, the compounds of the present invention can have effective effects on glucose regulation and / or circulating cholesterol levels, lowering circulating LDL levels and increasing HDL / LDL ratios. There is also. That is, the compounds of the present invention are direct or indirect treatments for any condition caused or characterized by overweight, such as obesity, pathological obesity, obesity-related inflammation, obesity-related bile sac. It can be used to treat and / or prevent diseases, obesity-induced sleep apnea. These are also poorly controlled blood glucose, or dyslipidemia (eg, elevated LDL levels or decreased HDL / LDL ratios), diabetes (especially type 2 diabetes), metabolic syndrome, hypertension, atherosclerosis, atherosclerosis. It may also be used to prevent symptoms caused or characterized by sclerosis, arteriosclerosis, coronary heart disease, peripheral arterial disease, stroke or microvascular disease. The effects of the present invention on these symptoms may be the result of the effects on the symptoms on body weight, or they may be independent.

The present invention also provides the compounds of the present invention for use in medical therapeutic methods, in particular methods of treating symptoms as described above.

The present invention also provides the use of the compounds of the present invention in the preparation of a medicament for the treatment of such symptoms.

The compounds of the present invention can be administered as part of combination therapy with agents for the treatment of diabetes, obesity, dyslipidemia, or hypertension.

In such cases, these two active substances may be administered together or separately, as part of the same pharmaceutical formulation or as separate formulations.

Thus, the compounds of the invention include, but are not limited to, biguanides (eg metformin), sulfonylureas, meglitinide or glinide (eg nateglinide), DPP-IV inhibitors, glitazones, SGLT2 inhibitors, insulin or insulin analogs. Can be used in combination with drugs. Examples of insulin analogs include Lantus ™, Novorapid ™, Humalog ™, Novomix ™, Actraphane HM ™, Levemir ™, and Apidra ™. Not limited.

The compound also includes glucagon-like peptide receptor 1 agonist, peptide YY or its analogs, cannabinoid receptor 1 antagonist, lipase inhibitor, melanocortin receptor 4 agonist, melanin-enriching hormone receptor 1 antagonist, fentermin (alone, Antis including, but not limited to, norepinephrine / dopamine reuptake inhibitors in combination with opioid receptor antagonists (eg, in combination with bupropion and naltrexone), or serotonin agonists (eg, lorcaserin). Can be used in combination with obesity drugs.

The compound can also be used in combination with antihypertensive agents including, but not limited to, angiotensin converting enzyme inhibitors, angiotensin II receptor blockers, diuretics, beta blockers, or calcium channel blockers.

The compound can be used in combination with anti-dyslipidemia agents including, but not limited to, statins, fibrates, niacins, or cholesterol absorption inhibitors.

That is, the present invention further provides a composition or a therapeutic kit containing the compound of the present invention and, for example, the above-mentioned antidiabetic drug, antiobesity drug, antihypertensive drug, or antidyslipidemia drug. Also provided are such compositions or therapeutic kits for use in medical therapeutic methods, especially for the treatment of the above-mentioned symptoms.

The compound of the present invention can be produced by synthetic chemistry. Therefore, the present invention provides a method for synthesizing the compound of the present invention.

As described above, the present invention comprises a nucleic acid encoding a peptide sequence X or XZ, as well as an expression vector containing the aforementioned nucleic acid sequence (possibly operably linked to a sequence that directs its expression), and said nucleic acid. Alternatively, it extends to the host cell containing the expression vector. Preferably, the host cell is capable of expressing and optionally secreting the compounds of the invention.

The present invention provides a method for producing a compound of the present invention, which comprises culturing a host cell under conditions suitable for expressing the peptide sequence X or XZ and purifying the compound thus produced. To do. This is especially useful if the peptide contains only naturally occurring amino acids.

If the compounds of the invention contain one or more non-naturally occurring amino acids and / or residue Ψ, this method expresses a peptide sequence containing one or more differences from sequence X or XZ. And optionally purify the compound thus produced to add or modify (eg, chemically modify) one or more amino acids to obtain the compounds of the invention or compounds comprising the amino acid sequence X or XZ. May include manufacturing.

Whatever method is used to produce the compounds of the invention, the method sequences, in particular to introduce one or more lipophilic moieties, as specified elsewhere herein. It may include one or more additional steps to modify (eg, chemically modify) X or XZ.

The invention further provides host cells capable of expressing and optionally secreting the nucleic acids of the invention, expression vectors of the invention, or compounds of the invention for use in medical therapeutic methods. It will be appreciated that the nucleic acids, expression vectors, and host cells can be used to treat any of the disorders described herein that can be treated with the compounds of the invention. Thus, when referred to as a Therapeutic composition comprising a compound of the invention, administration of a compound of the invention, or any therapeutic use thereof, unless otherwise indicated, the nucleic acid, expression vector, or host of the invention. It should be construed to include equivalent use of cells.

Throughout this specification, the conventional one-letter and three-letter codes are used for natural amino acids, and other amino acids such as D-Ala or DAla (D-alanine), Aib (α-aminoisobutyric acid), Orn (ornithine) ), NMeSer or N-Me-Ser (N-methylserine), Ac3c (1-amino-cyclopropanecarboxylic acid), Ac4c (1-amino-cyclobutanecarboxylic acid), Ac5c (1-amino-cyclopentanecarboxylic acid), Abu ((S) -2-aminobutyric acid) uses commonly accepted abbreviations.

Ac3c, Ac4c, and Ac5c have similar structures and are interchangeable to some extent, but Ac4c may be preferred.

Glucagon is a 29 amino acid peptide having a sequence corresponding to amino acid positions 53 to 81 of preproglucagon, and has the sequence His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-. It has Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr. Oxintmodulin (OXM) is the carboxy-terminal extension of the octapeptide (at amino acids 82-89 of the preproglucagon called "intervening peptide 1" or IP-1 and is in the sequence Lys-Arg-Asn-Arg-Asn-Asn- 37 amino acid peptides containing the complete 29 amino acid sequences of (with Ile-Ala) and glucagon; that is, the entire sequence of human oxintomodulin is His-Ser-Gln-Gly-Thr-Phe-Thr. -Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-Lys-Arg-Asn -Arg-Asn-Asn-Ile-Ala. The major bioactive fragment of GLP-1 is produced as a C-terminal amidated peptide of 30 amino acids corresponding to amino acids 98-127 of preproglucagon.

That is, the term "natural glucagon" is the sequence H-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala- Refers to a natural human glucagon with Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-OH.

It is conceivable that the amino acids in the sequence X of the compound of the present invention are numbered consecutively from 1 to 29 from the usual N-terminal toward the C-terminal. Therefore, when we refer to the "position" within X, it should be understood that it is similar to the position within natural human glucagon and other molecules.

The compounds of the present invention are enzymatically used, for example, to stabilize the conformation and / or secondary structure of glucagon analog peptides and / or as described in WO 99/46283. To make the glucagon analog peptide more resistant to hydrolysis, it may contain the C-terminal peptide sequence Z of 1-20 amino acids.

If present, Z represents a peptide sequence of 1 to 20 amino acid residues, eg, in the range of 1 to 15, more preferably in the range of 1 to 10, especially 1 to 7 amino acid residues. Within the range of, eg, 1, 2, 3, 4, 5, 6, or 7 amino acid residues, eg, within the range of 6 amino acid residues. Each of the amino acid residues in the peptide sequence Z is Ala, Leu, Ser, Thr, Tyr, Cys, Glu, Lys, Arg, Dbu (2,4-diaminobutyric acid), Dpr (2,3-diaminopropanoic acid). , And Ornithine can be selected independently. Preferably the amino acid residue is selected from Ser, Thr, Tyr, Glu, Lys, Arg, Dbu, Dpr, and Orn, and more preferably only from Glu, Lys, and Cys. The amino acids can have a D- or L-orientation and, in certain embodiments, have an L-orientation. A particularly suitable sequence Z is a sequence of 4, 5, 6, or 7 contiguous lysine residues (ie, Lys ₃ , Lys ₄ , Lys ₅ , Lys ₆ , or Lys ₇ ), particularly 5 or 6. Is a sequence of consecutive lysine residues. Other typical sequences of Z are described in WO 01/04156 Pamphlet. Alternatively, the C-terminal residue of sequence Z may be a Cys residue. This can help modify the compound (eg, PEGylation, or binding to albumin). In such embodiments, the sequence Z may be, for example, only one amino acid in length (ie Z = Cys), or may be 2, 3, 4, 5, 6 or even more amino acids in length. .. The other amino acids therefore act as spacers between peptide X and the terminal Cys residue.

Peptide sequence Z has no more than 25% sequence identity with the corresponding sequence of the IP-1 portion of human OXM, which has the sequence Lys-Arg-Asn-Arg-Asn-Asn-Ile-Ala.

The "% (%) amino acid sequence identity" of one peptide or polypeptide sequence to another polypeptide sequence (eg IP-1) aligns the two with each other and, if necessary, for optimal alignment. When a gap is introduced, it is calculated as a percentage of the number of amino acid residues in one polypeptide sequence that are identical to the corresponding positions in the corresponding sequence of another polypeptide. % Identity values can be measured using WU-BLAST-2 (Altschul et al., Methods in Enzymology, 266: 460-480 (1996)). WU-BLAST-2 uses several search parameters, most of which are set to default values. Adjustable parameters are set to the following values: overlap span = 1, overlap fraction = 0.125, word threshold (T) = 11. The value of% amino acid sequence identity is determined by dividing the number of matching identical residues determined by WU-BLAST by the total number of residues in the reference sequence and multiplying by 100 (maximizing the alignment score). In order to do so, the gap introduced into the reference sequence by WU-BLAST-2 is ignored).

That is, when Z is optimally aligned with the eight amino acids of IP-1, there are no more than two amino acids that are identical to the corresponding amino acids of IP-1.

In certain embodiments, Z is absent.

When the compound of the present invention contains a residue Ψ, the Ψ contains a residue Lys, Arg, Orn or Cys whose side chain is attached to a lipophilic substituent. Lys may be preferred. The lipophilic substituent may be covalently attached to an atom in the amino acid side chain or, optionally, attached to the amino acid side chain by a spacer.

Without being bound by any particular theory, lipophilic substituents bind to plasma proteins in the bloodstream (eg, albumin) to block the compounds of the invention from enzymatic degradation, which results in the half-life of the compounds. To raise. It is also possible to regulate the efficacy of the compound, for example on the glucagon receptor and / or the GLP-1 receptor.

In certain embodiments, only one amino acid side chain is attached to a lipophilic substituent. In another embodiment, the two amino acid side chains are each attached to a lipophilic substituent. In yet another embodiment, three or more amino acid side chains are each attached to a lipophilic substituent. When the compound contains two or more lipophilic substituents, these substituents may be the same or different.

The lipophilic substituent contains or consists of a lipophilic moiety Z ¹ , which may be covalently attached directly to an atom in the amino acid side chain, or optionally attached to the amino acid side chain by ^{spacer Z 2.} You may.

The term "conjugated" is used herein to describe the physical attachment of one identifiable chemical part to another, and the structural relationship between such parts. .. It should not be considered to refer to a particular synthetic method.

The lipophilic moiety may be attached to the amino acid side chain or spacer via an ester, sulfonyl ester, thioester, amide, carbamate, urea, or sulfonamide. Thus, preferably, the lipophilic substituent contains an acyl group, a sulfonyl group, an N atom, an O atom, or an S atom, which forms part of an ester, sulfonyl ester, thioester, amide, or sulfonamide. It will be understood to include. Preferably, the acyl group in the lipophilic substituent forms part of the amino acid side chain or spacer with the amide or ester.

The lipophilic moiety may contain hydrocarbon chains with 4-30 C atoms. Preferably, it has at least 8 or 12 C atoms, preferably 24 or less carbon atoms, or 20 or less carbon atoms. The hydrocarbon chain may be linear or branched and may be saturated or unsaturated. It is understood that the hydrocarbon chain is preferably substituted with a moiety that forms part of an amino acid side chain or bond to a spacer, such as an acyl group, a sulfonyl group, an N atom, an O atom or an S atom. Let's go. Most preferably, the hydrocarbon chain is substituted with an acyl group, so the hydrocarbon chain may be part of an alkanoyl group such as palmitoyl, caproyl, lauroyl, myristoyl, or stearoyl.

Therefore, the lipophilic moiety can have the following equation.

In the formula, A may be, for example, an acyl group, a sulfonyl group, NH, N-alkyl, O atom, or S atom, and is preferably an acyl group. n is an integer of 3 to 29, preferably an integer of 7 to 25, more preferably 11 to 21, and even more preferably an integer of 15 to 19.

Hydrocarbon chains can be further substituted. For example, this hydrocarbon chain can be replaced by up to three substituents selected from _{NH 2} , OH, and COOH, especially at the free end of the molecule distal to the spacer or peptide. For example, it may contain a free carboxylic acid group.

If the hydrocarbon chain is further substituted, it is preferably further substituted with only one substituent. Alternatively or additionally, the hydrocarbon chain may include, for example, the cycloalkanes or heterocycloalkanes shown below.

Preferably, the cycloalkane or heterocycloalkane is a 6-membered ring. Most preferably, it is piperidine.

Alternatively, the lipophilic moiety may be based on the cyclopentanophenatorene backbone, which may be partially or completely unsaturated or saturated. The carbon atoms in the backbone can be replaced with Me or OH, respectively. For example, the lipophilic substituent may be choryl, deoxycholyl, or lithocholyl.

As described above, the lipophilic moiety may be bound to the amino acid side chain by a spacer. If present, the spacer is attached to the lipophilic moiety and the amino acid side chain. The spacer may be independently attached to the lipophilic moiety and the amino acid side chain by an ester, sulfonyl ester, thioester, amide, carbamate, urea, or sulfonamide. Therefore, it may contain two moieties that are independently selected from the acyl, sulfonyl, N, O, or S atoms. The spacer may have the following equation.

In the formula, B and D are selected independently of the acyl, sulfonyl, NH, N-alkyl, O and S atoms, respectively, preferably of acyl and NH, respectively. Preferably n is 1 to 10 integers, preferably 1 to 5 integers. The spacer may be further substituted with one or more substituents selected from _{C 0-6} alkyl, C _0-6 alkyl amine, C _0-6 alkyl hydroxy, and C _{0-6 alkyl carboxy.}

Alternatively, the spacer may have two or more repeating units of the above formula. B, D and n are each independently selected for each repeating unit. Adjacent repeating units may be covalently bonded to each other via parts B and D, respectively. For example, the B and D moieties of adjacent repeating units may together form an ester, sulfonyl ester, thioester, amide, or sulfonamide. As mentioned above, the free B and D units at each end of the spacer are attached to the amino acid side chain and the lipophilic moiety.

Preferably, the spacer has 5 or less, 4 or less, or 3 or less repeating units. Most preferably, the spacer has two repeating units or is a single unit.

The spacer (if it has a repeating unit, one or more repeating units of the spacer) may be, for example, a natural or non-natural amino acid. It will also be appreciated that for amino acids with functionalized side chains, B and / or D may be a portion within the side chain of the amino acid. The spacer may be any natural or non-natural amino acid. For example, a spacer (or one or more repeating units of a spacer if it has a repeating unit) can be Gly, Pro, Ala, Val, Leu, Ile, Met, Cys, Phe, Tyr, Trp, His, Lys. , Arg, Gln, Asn, α-Glu, β-Glu, γ-GLu, Asp, Ser, Thr, Gaba, Aib, β-Ala, 5-aminopentanoyl, 6-aminohexanoyl, 7-aminoheptanoyl , 8-Amino Octanoyl, 9-Amin Nonanoyl, or 10-Amino Decanoyl.

For example, the spacer may be a single amino acid selected from γ-Glu, Gaba, β-Ala, and α-Glu.

The amino acids in the spacer having a stereocenter may be racemic, enantioenriched, or enantiopure. In some embodiments, each amino acid in the spacer is an independently L-amino acid. In some embodiments, each amino acid is an independent D-amino acid.

An example of a lipophilic substituent containing a lipophilic moiety is shown in the following formula.

Here, the Lys residue in the compound of the present invention is covalently bonded to γ-Glu (spacer) via an amide moiety. Palmitoyl (ie, hexadecanoyl) forms a hexadecanoyl-isoGLu group by covalently bonding to a γ-Glu spacer via an amide moiety.

This group may be present as Ψ in any compound of the invention.

Alternatively or additionally, one or more amino acid side chains in the compounds of the invention increase solubility and / or in vivo half-life (eg in plasma) and / or bioavailability. May be attached to the polymer moiety. Such modifications are also known to reduce therapeutic protein and peptide clearance (eg, renal clearance).

Those skilled in the art will use the general synthetic methods described in, for example, “Comprehensive Organic Transformations, A Guide to Functional Group Preparations”, 2nd edition, Larock, RC; Wiley-VCH: New York, 1999, with spacers. You will be aware of suitable techniques that can be used to carry out binding reactions with lipophilic moieties. Such a transformation can be performed at any suitable stage during the synthesis process.

The polymer moiety is preferably water soluble (amphipathic or hydrophilic), nontoxic, and pharmacologically inert. Suitable polymer moieties include polyethylene glycol (PEG), homopolymers or copolymers of PEG, monomethyl-substituted polymers of PEG (mPEG), polyoxyethylene glycerol (POG) and the like. See, for example, Int. J. Hematology 68: 1 (1998); Bioconjugate Chem. 6: 150 (1995); and Crit. Rev. Therap. Drug Carrier Sys. 9: 249 (1992).

Other suitable polymeric moieties include polyamino acids such as polylysine, polyaspartic acid, and polyglutamic acid (eg, Gombotz, et al. (1995), Bioconjugate Chem., Vol. 6: 332-351; Hudecz, et al. (1992), Bioconjugate Chem., Vol. 3, 49-57; Tsukada, et al. (1984), J. Natl. Cancer Inst., Vol 73 ,: 721-729; and Pratesi, et al. (1985), Br. J. Cancer, vol. 52: 841-848).

The polymer moiety may be straight or branched. It may have a molecular weight of 500-40,000 Da, for example 500-10,000 Da, 1000-5000 Da, 10,000-20,000 Da, 20,000-40,000 Da.

The compounds of the present invention may contain a plurality of such moieties, in which case the total molecular weight of such moieties will generally fall within the above range.

The polymer moiety can be attached (by covalent bond) to an amino, carboxyl, or thiol group on the amino acid side chain. Suitable examples are the thiol group of the Cys residue and the epsilon amino group of the Lys residue. Carboxyl groups of Asp and Glu residues can also be used.

Those skilled in the art will be familiar with the appropriate techniques that can be used to carry out the binding reaction. For example, the PEG moiety having a methoxy group can be attached to a Cys thiol group by a maleimide bond using a reagent commercially available from Nektar Therapeutics. See International Publication No. 2008/101017 Pamphlet and the above cited references for appropriate chemical details.

Peptide Synthesis The compounds of the present invention can be prepared by standard synthetic methods, recombinant expression systems, or any other state-of-the-art technique. That is, the glucagon analog can be synthesized by many methods, for example, a method including the following steps.
(A) Synthesizing peptides using solid phase or liquid phase methods, either stepwise or by fragment assembly, and isolating and purifying the final peptide product, or
(B) Expressing a nucleic acid construct encoding a peptide in a host cell and recovering the expression product from the host cell or culture medium, or
(C) Cell-free in vitro expression of the nucleic acid construct encoding the peptide and recovery of the expression product, or
Obtaining a peptide fragment by any combination of methods (a), (b), and (c), then ligating the fragments to obtain a peptide, and recovering the peptide.

The analogs of the present invention are preferably synthesized by solid phase or liquid phase peptide synthesis. See International Publication No. 98/11125 and Fields, GB et al., 2002, “Principles and practice of solid-phase peptide synthesis” in Synthetic Peptides, 2nd edition, and examples in it. I want to be.

For recombinant expression, the nucleic acid fragments of the invention are usually inserted into a suitable vector to form a cloning or expression vector carrying the nucleic acid fragments of the invention. Such novel vectors are also part of the present invention. The vector can be in the form of a plasmid, phage, cosmid, minichromosome, or virus, depending on the purpose and type of application, but bare DNA, which is only transiently expressed in a particular cell, is an important vector. is there. Suitable cloning and expression vectors of the invention (plasmid vectors) are self-replicating, thereby allowing high levels of expression or high copy counts for subsequent cloning.

In general, expression vectors are promoters that drive the expression of nucleic acid fragments of the invention in the 5'→ 3'direction and in activable linkages; optionally secreted (extracellular or, if possible, intraperiplasmic). Includes a nucleic acid sequence encoding a leader peptide that enables (secretion to); a nucleic acid fragment encoding a peptide of the invention; optionally a nucleic acid sequence encoding a terminator. These vectors may include additional features such as selectable markers and origins of replication. When manipulating with an expression vector in a production or cell line, it may be preferable that the vector be able to integrate into the genome of the host cell. Those skilled in the art will be familiar with the appropriate vectors and can design according to their specific requirements.

The vectors of the invention are used to transform host cells for producing the peptides of the invention. Such transformed cells, which are part of the invention, are used for the growth of nucleic acid fragments and vectors of the invention, or for the production of recombinants of the precursor peptides of the invention. Can be a cultured cell or cell line to be produced.

Suitable transformed cells of the present invention are bacteria [eg, E. coli, Bacillus subtilis, Salmonella], or Mycobacterium. Bacteria such as species (preferably non-pathogenic, such as M-Bobis BSG), yeast (eg, Saccharomyces cerevisiae, and Pichia pastoris), and protozoa, or traits. The transformed cell can be obtained from a multicellular organism, i.e. it may be a fungal cell, an insect cell, an algae cell, a plant cell, or an animal cell (eg, a mammalian cell). For cloning and / or optimized expression. For the purposes, it is preferred that the transformed cell is capable of replicating the nucleic acid fragment of the invention. A cell expressing the nucleic acid fragment is a useful embodiment of the invention, small or large of the peptides of the invention. Can be used for scale preparation.

When the peptide of the present invention is produced using transformed cells, it is convenient that the expression product thereof is secreted into a culture medium, although it is not essential.

efficacy
Binding of the corresponding compound to the GLP-1 or glucagon (Glu) receptor can be used as an indicator of agonist activity, but generally measures intracellular signaling triggered when the compound binds to the corresponding receptor. It is preferable to use a biological assay. For example, activation of glucagon receptors by glucagon agonists will stimulate the production of cellular cyclic AMP (cAMP). Similarly, activation of the GLP-1 receptor by a GLP-1 agonist will stimulate the production of cAMP in the cell. That is, the production of cAMP in the appropriate cells expressing one of these two receptors can be utilized to monitor the activity of the corresponding receptor. Therefore, it is possible to measure agonist activity against both types of receptors by appropriately using a pair of cells of a type that expresses one receptor each and does not express the other receptor.

Those skilled in the art will be familiar with the appropriate assay format, but will be exemplified below. The GLP-1 and / or glucagon receptors can have a receptor sequence as described in the Examples. For example, the assay should use a human glucagon receptor (Glucagon-R) with primary accession number GI: 4503947 and / or a human glucagon-like peptide 1 receptor (GLP-1R) with primary accession number GI: 166795283. (When referring to the sequence of the precursor protein, it should be understood, of course, that the assay may use a mature protein lacking the signal sequence).

EC ₅₀ can be used as a numerical measure of agonist efficacy at a receptor. The EC ₅₀ values are, in one particular assay is a measure of the concentration of compound required to achieve half-maximal activity of the compound. That is, for example, in certain assay, compounds with low EC ₅₀ [GLP-1] from the EC ₅₀ [GLP-1] for glucagon, can be considered to have a potency of greater than glucagon GLP-1 receptor agonists ..

The compounds described herein are typically GluGLP-1 dual agonists, as determined by the observation that both glucagon and GLP-1 receptors can stimulate the production of cAMP. Stimulation of each receptor can be measured in an independent assay and then compared to each other.

By comparing The EC ₅₀ values for GLP-1 receptor _{(EC 50 [GLP-1-} R]) EC 50 values for the glucagon receptor (EC ₅₀ [GlucagonR]), relative to a certain compound GLP- The 1R selectivity can be calculated by the following formula.
Relative GLP-1R selectivity [Compound] = (EC ₅₀ [GLP-1-R]) / (EC ₅₀ [Glucagon-R])

The term "EC ₅₀ " typically means a concentration that represents 50% of the maximum response of a particular marker level to a particular receptor or to receptor function and is inhibited or inhibited by a particular biochemical context. May refer to antagonist activity.

Without being bound by any particular theory, the relative selectivity of one compound to directly compare its potency on the GLP-1 or glucagon receptor with its potency on other receptors. May be possible. For example, the higher the relative GLP-1 selectivity of a compound, the more effective the compound may be on the GLP-1 receptor than on the glucagon receptor. Typically, this result is a glucagon receptor and GLP-1 receptor from the same species (eg, human glucagon receptor and human GLP-1 receptor, or mouse glucagon receptor and mouse GLP-1 receptor). Compare between.

For specific levels of glucagon-R agonist activity, the compounds of the present invention are capable of exhibiting higher levels of GLP-1R agonist activity (ie, greater potency at the GLP-1 receptor) than glucagon. Compounds can have higher relative GLP-1R selectivity than human glucagon. The absolute potency of a particular compound on glucagon and GLP-1 receptors may be higher, lower, or higher than that of natural human glucagon, as long as the relative GLP-1R selectivity is appropriate. It will be understood that they may be approximately equal.

However the compounds of the present invention may have a lower EC ₅₀ [GLP-1R] than human glucagon. The compounds are less than 10 times than the EC ₅₀ [Glucagon -R] human glucagon high, twice than EC ₅₀ for human glucagon [glucagon -R] less than 5 times higher than, or EC ₅₀ of human glucagon [Glucagon -R] less high, while maintaining the EC ₅₀ [glucagon -R], it may have a lower _{EC 50 [GLP-1-R} ] than glucagon.

The compounds of the present invention may have a EC ₅₀ [Glucagon -R] is less than 2 times greater than EC ₅₀ [Glucagon -R] of human glucagon. The compounds, EC ₅₀ of less than 2 times the EC ₅₀ [Glucagon -R] human glucagon has the glucagon -R], and, EC ₅₀ of less than half of the _{EC 50 [GLP-1-R} ] of the human glucagon [GLP-1-R], EC 50 of less than 1 times the 5 minute _{EC 50 [GLP-1-R} ] of the human glucagon [GLP-1-R] or human glucagon _{EC 50, [GLP-1-} R ] May have less than 1/10 EC ₅₀ [GLP-1-R].

The relative GLP-1R selectivity of this compound may be 0.05 to 20. For example, the compounds are 0.05-0.20, 0.1-0.30, 0.2-0.5, 0.3-0.7, or 0.5-1.0; 1.0-2.0, 1.5-3.0, 2.0-4.0, or 2.5-5.0; or 0.05-20, 0.075. It may have a relative selectivity of ~ 15, 0.1 ~ 10, 0.15 ~ 5, 0.75 ~ 2.5, or 0.9 ~ 1.1.

In some embodiments, EC ₅₀ of both glucagon -R and GLP-1R, for example, for the human glucagon receptor and the human GLP-1 receptor, certain compounds, it may be desirable less than 1 nM.

Therapeutic Applications The compounds of the present invention can provide attractive therapeutic and / or prophylactic options, especially for metabolic disorders, including obesity and diabetes, as described below.

Diabetes includes a group of metabolic disorders characterized by hyperglycemia due to deficiencies in insulin secretion, insulin action, or both. Acute signs of diabetes include excessive urine production, consequent increased thirst and fluid intake, visual impairment, unexplained weight loss, malaise, and altered energy metabolism. Chronic hyperglycemia in diabetes is associated with long-term damage, dysfunction, and dysfunction of various organs, especially the eyes, kidneys, nerves, heart and blood vessels. Diabetes is classified into type 1 diabetes, type 2 diabetes, and gestational diabetes based on the etiologic characteristics.

Type 1 diabetes accounts for 5-10% of all diabetic cases and is caused by the autoimmune destruction of insulin-secreting pancreatic β-cells.

Type 2 diabetes accounts for 90% to 95% of diabetic cases and occurs as a result of a complex combination of metabolic disorders. Type 2 diabetes results from inadequate endogenous insulin production and the inability to maintain plasma glucose levels below the diagnostic threshold.

Gestational diabetes refers to any degree of impaired glucose tolerance seen during pregnancy.

Prediabetes refers to a condition that includes fasting glycemic disorders and impaired glucose tolerance, which occurs when blood glucose levels are elevated but below the levels that make a clinical diagnosis of diabetes.

Most people with type 2 diabetes and prediabetes have abdominal obesity (excessive adipose tissue around the abdominal viscera), atherosclerosis (high triglyceride, low HDL cholesterol that promotes plaque accumulation in the arterial wall). , And / or blood fat disorders including high LDL cholesterol), elevated blood pressure (high blood pressure), pre-thrombotic status (eg, elevated blood fibrinogen or plasminogen activator inhibitor 1), and pre-inflammatory status The high frequency of additional metabolic risk factors, including (eg, elevated C-reactive proteins in the blood), increases the risk of morbidity and mortality.

Obesity, on the other hand, increases the risk of developing prediabetes, type 2 diabetes, and certain types of cancer, obstructive sleep apnea, and gallbladder braider disease.

Dyslipidemia is associated with an increased risk of cardiovascular disease. HDL is clinically important because there is an inverse correlation between plasma high-density lipoprotein (HDL) levels and the risk of atherosclerosis. High levels of LDL are closely associated with atherosclerosis, as most of the cholesterol stored in atherosclerotic plaques is derived from low-density lipoprotein (LDL). The HDL / LDL ratio is an indicator of the clinical risk of atherosclerosis, especially coronary atherosclerosis.

Metabolic syndrome is characterized by a person having a group of metabolic risk factors. These include abdominal obesity (excessive adipose tissue around the abdominal viscera), atherosclerotic dyslipidemia (promoting plaque accumulation in the arterial wall, high triglyceride, low HDL cholesterol, and / or high LDL cholesterol in the blood. Dyslipidemia), elevated blood pressure (hypertension), insulin resistance and abnormal glucose tolerance, prethrombogenic status (eg, elevated fibrinogen or plasminogen activator inhibitor 1 in the blood), and preinflammatory status (eg, elevated) (Elevation of C-reactive protein in blood) is included.

Individuals with metabolic syndrome are at increased risk of other diseases associated with coronary heart disease and other symptoms of arteriosclerosis (eg, stroke and peripheral vascular disease). The major risk factor for this syndrome appears to be abdominal obesity.

Without being bound by any particular theory, the compounds of the invention are believed to act as dual agonists for both human glucagon and human GLP1 receptors (abbreviated herein as dual GluGLP-1 agonists). To do). A dual agonist is a combination of, for example, the effect of glucagon on fat metabolism and, for example, the effect of GLP-1 on blood glucose levels and food intake. Thus, they may have the effect of accelerating the removal of excess adipose tissue, inducing sustained weight loss and improving glycemic control. Double GluGLP-1 agonists may also act to reduce cardiovascular risk factors such as high cholesterol, high LDL cholesterol, and low HDL / LDL cholesterol ratios.

Thus, the compounds of the present invention prevent weight gain, promote weight loss, reduce excess weight, or obesity (including pathological obesity) and related diseases and health in subjects in need thereof. Conditions (including, but not limited to, obesity-related inflammation, obesity-related bile sac disease, and obesity-induced sleep aspiration) (eg, appetite, feeding, food intake, calorie intake, and / or energy consumption) Can be used as a therapeutic agent (under the control of). The compounds of the present invention also provide metabolic syndrome, insulin resistance, impaired glucose tolerance, prediabetes, increased fasting glucose, type 2 diabetes, hypertension, atherosclerosis, arteriosclerosis in subjects in need of treatment. It can also be used to treat symptoms caused by or associated with glucose dysregulation, including disease, coronary heart disease, peripheral arterial disease, and stroke. Some of these conditions may be associated with obesity. However, the effects of the compounds of the invention on these conditions may be wholly or partially intervening or independent of their effects on body weight.

The synergistic effect of dual GluGLP-1 agonists may result in a reduction in cardiovascular risk factors such as high cholesterol and LDL, which may be completely independent of their effect on body weight.

Thus, as described above, the present invention provides the use of the compounds of the present invention in the treatment of the above-mentioned symptoms in individuals in need thereof.

The present invention also provides the compounds of the present invention for use in medical therapeutic methods, in particular methods of treating symptoms as described above.

In a preferred embodiment, the described compounds can be used to treat diabetes, especially type 2 diabetes.

In certain embodiments, the present invention includes the use of compounds for treating diabetes, especially type 2 diabetes, in individuals in need thereof.

In a preferred embodiment, the described compounds can be used to prevent weight gain or promote weight loss.

In certain embodiments, the invention comprises the use of a compound to prevent weight gain or promote weight loss in an individual in need thereof.

In certain embodiments, the present invention provides treatments for symptoms caused or characterized by overweight in individuals in need thereof, such as obesity, morbid obesity, preoperative morbid obesity, obesity. Inflammation associated with obesity, obesity-related biliary sac disease, obesity-induced sleep aspiration, pre-diabetic disease, diabetes, especially type 2 diabetes, hypertension, atherosclerosis, atherosclerosis, arteriosclerosis, coronary artery Includes the use of compounds in the treatment and / or prevention of sexual heart disease, peripheral arterial disease, stroke or microvascular disease.

In another embodiment, the described compounds can be used in a manner that lowers circulating LDL levels and / or raises the HDL / LDL ratio.

In certain embodiments, the invention comprises the use of a compound in a method of lowering circulating LDL levels and / or increasing HDL / LDL ratios in individuals in need thereof.

In another embodiment, the described compounds can be used in a manner that lowers circulating triglyceride levels.

Pharmaceutical Compositions The compounds of the present invention can be formulated as pharmaceutical compositions prepared for storage or administration. Such compositions typically contain a therapeutically effective amount of a compound of the invention in a pharmaceutically acceptable carrier in a suitable form.

The therapeutically effective amount of a compound of the invention will depend on the route of administration, the type of mammal being treated, and the particular physical characteristics of the mammal under consideration. These relationships in determining these factors and quantities are well understood by those skilled in the medical arts. This amount and method of administration can be adjusted to achieve optimal efficacy and may depend on body weight, diet, concomitant medications, and other factors (those skilled in the medical art are familiar with). The most appropriate dosing size and dosing regimen for human use may be derived from the results obtained by the present invention or may be confirmed in well-planned clinical trials. The compounds of the present invention may be particularly useful for the treatment of humans.

Effective dosage and treatment protocols can be determined by conventional methods starting at low doses in laboratory animals, increasing doses while tracking efficacy, and also altering the overall dosing regimen. .. Clinicians can consider many factors in determining the optimal dose for a subject. Such considerations are known to those of skill in the art.

The term "pharmaceutically acceptable carrier" includes any standard pharmaceutical carrier. Pharmaceutically acceptable carriers for therapeutic use are known in the pharmaceutical field and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A.R. Gennaro edit. 1985). For example, sterile pH or physiological pH saline and phosphate buffered saline can be used. pH buffers include phosphate, citrate, acetate, tris (hydroxymethyl) aminomethane (TRIS), N-tris (hydroxymethyl) methyl-3-aminopropanesulfonic acid (TAPS), ammonium bicarbonate, It may be diethanolamine, histidine (which is a suitable buffer), arginine, lysine, or acetate, or a mixture thereof. The term further includes any drug described in the United States Pharmacopeia for use in animals, including humans.

The term "pharmaceutically acceptable salt" refers to any salt of any of the compounds of the invention. Salts include pharmaceutically acceptable salts such as acid addition salts and basic salts. Examples of acid addition salts include hydrochlorides, citrates, and acetates. As an example of a basic salt, cations are alkali metals such as sodium and potassium, calcium and ammonium ions ⁺ N (R ³ ) ₃ (R ⁴ ) (in the formula, R ³ and R ⁴ are optionally substituted Salts selected from alkaline earth metals such as C _1-6 alkyl, optionally substituted C _2-6 alkenyl, optionally substituted aryl, optionally substituted heteroaryl) Can be mentioned. Other examples of pharmaceutically acceptable salts are “Remington's Pharmaceutical Sciences”, 17th edition. Ed. Alfonso R. Gennaro (Ed.), Mark Publishing Company, Easton, PA, USA, 1985, and more recent editions. , And the Encyclopaedia of Pharmaceutical Technology.

"Treatment" is an approach to obtain beneficial or desired clinical results. For the purposes of the present invention, beneficial or desired clinical outcomes include, but are not limited to, symptomatology relief, disease severity reduction, disease state stabilization (ie, non-exacerbation) conditions, disease. It includes delaying or slowing the progression, improving or alleviating the disease state, and remission (partial or wholly), which may be detectable or undetectable. "Treatment" may also mean prolonging survival compared to the expected survival without treatment. "Treatment" is an intervention intended to prevent the development of a disorder or alter the condition of the disorder. Thus, "treatment" refers to both therapeutic treatment and prognostic or prophylactic measures in certain embodiments. Those in need of treatment include those who already have a disability and those whose disability should be prevented. Treatment means inhibiting or reducing the spread of the condition or symptoms (eg, weight gain, hyperglycemia) compared to when no treatment is present, and does not necessarily mean complete cessation of the associated symptoms. Absent.

The pharmaceutical composition may be in a unit dosage form. In such dosage forms, the composition is divided into unit doses containing the appropriate amount of active ingredient. Unit dosage forms may be packaged preparations containing individual amounts of preparations, such as packetized tablets, capsules, and powders in vials or ampoules. The unit dosage form may also be itself a capsule, cachet, or tablet, or may be in the form of a package of an appropriate number of these agents. It may be provided in the form of a single dose injection, eg in the form of a pen. In certain embodiments, the form of the package may include a label or insert with instructions for use. The composition can be formulated for any suitable route of administration and means of administration. Pharmaceutically acceptable carriers or diluents are oral, rectal, nasal, topical (including oral and sublingual), vaginal, or parenteral (subcutaneous, intramuscular, intravenous, intradermal, and trans). Includes those used in preparations suitable for administration (including skin). This preparation is conveniently provided in a unit dosage form and can be prepared by any method known in the field of pharmaceutics.

Subcutaneous or transdermal administration may be particularly suitable for the compounds described herein.

The compositions of the present invention are further known to those skilled in the art for time therapy to enhance the stability of the compounds, to increase their bioavailability, to increase their solubility, and to reduce their side effects. To achieve, and to increase patient compliance, or for any combination thereof, for example, through covalent bonds, hydrophobic and electrostatic interactions, drug carriers, drug delivery systems, and It can be formulated or combined with an evolved drug delivery system. Examples of carriers, drug delivery systems, and evolved drug delivery systems are, but are not limited to, polymers such as cellulose and derivatives, polysaccharides such as dextran and derivatives, starch and derivatives, poly (vinyl alcohol), acrylates and Methacrylate polymers, polylactic acid and polyglycolic acid, and their block copolymers, polyethylene glycols, carrier proteins such as albumin, gels such as thermal gelling systems, such as block copolymers known to those of skill in the art, micelles, liposomes, microspheres. , Nanoparticles, liquid crystals and their dispersions, L2 phase and its dispersions known to those skilled in the art for liquid-aqueous phase behavior, polymer micelles, polyemulsified emulsions, self-emulsifying, self-microemulsifying, cyclodextrin and itss. Includes derivatives and dendrimers.

Combination Therapies The compounds or compositions of the invention may be administered as part of combination therapies with agents for the treatment of obesity, hypertension, dyslipidemia, or diabetes.

In such cases, these two active substances may be administered together or separately, as part of the same pharmaceutical formulation or as separate formulations.

That is, the compounds or compositions of the present invention are, but are not limited to, glucagon-like peptide receptor 1 agonist, peptide YY or its analogs, cannabinoid receptor 1 antagonist, lipase inhibitor, melanocortin receptor 4 agonist, melanin. Aggregate hormone receptor 1 antagonist, fentermin (alone or in combination with topiramate), norepinephrine / dopamine reuptake inhibitor in combination with opioid receptor antagonist (eg, in combination with bupropion and naltrexone), or serotonin agonist It can be used in combination with anti-obesity agents including (eg, lolcaserin).

The compounds or compositions of the invention are in combination with, but not limited to, antihypertensive agents including angiotensin converting enzyme inhibitors, angiotensin II receptor blockers, diuretics, beta blockers, or calcium channel blockers. Can be used.

The compounds or compositions of the present invention can be used in combination with, but are not limited to, dyslipidemia agents including statins, fibrates, niacin, and / or cholesterol absorption inhibitors.

Furthermore, the compounds or compositions of the invention are different, but not limited to, biguanides (eg, metformin), sulfonylureas, meglycinides or glinides (eg, nateglinide), DPP-IV inhibitors, SGLT2 inhibitors, glitazones. It can be used in combination with antidiabetic drugs, including GLP-1 agonists, insulin or insulin analogs. In a preferred embodiment, the compound or salt thereof is used in combination with insulin or an insulin analog, a DPP-IV inhibitor, sulfonylurea or metformin, in particular sulfonylurea or metformin, to achieve proper glycemic control. .. Examples of insulin analogs include, but are not limited to, Lantus, Novorapid, Humalog, Novomix, and Actrafan HM, Levemil, and Apidola.

Example 1
Solid-phase peptide synthesis (SPPS) was performed in NMP on polystyrene resin (TentaGel S Ram) using a microwave-assisted synthesizer using the general synthetic standard Fmoc method for glucagon analogs. HATU was used as a binding reagent with DIPEA as a base. Piperidine (20% in NMP) was used for deprotection. Pseudoproline: Fmoc-Phe-Thr (psiMe, Mepro) -OH and Fmoc-Asp-Ser (psiMe, Mepro) -OH (purchased from NovaBiochem) were used where appropriate.

The following abbreviations were used.
Boc: tert-butyloxycarbonyl
ivDde: 1- (4,4-dimethyl-2,6-dioxocyclohexylidene) 3-methyl-butyl
Dde: 1- (4,4-dimethyl-2,6-dioxocyclohexylidene) -ethyl
DCM: Dichloromethane
DMF: N, N-dimethylformamide
DIPEA: Diisopropylethylamine
EDT: 1,2-Ethaneditor
EtOH: Ethanol
Et ₂ O: Diethyl ether
HATU: N-[(dimethylamino) -1H-1,2,3-triazole [4,5-b] pyridin-1-ylmethylene] -N-methylmethanamineium hexafluorophosphate N-oxide
MeCN: acetonitrile
NMP: N-methylpyrrolidone
TFA: Trifluoroacetic acid
TIS: triisopropylsilane

Disconnection:
Crude peptides were cleaved from the resin by treatment with 95 / 2.5 / 2.5% (v / v) TFA / TIS / water at room temperature (rt) for 2 hours. Most of the TFA was removed under reduced pressure, the crude peptide was precipitated, washed with diethyl ether and dried at ambient temperature to constant weight.

The following compounds were synthesized.

Compound number
1 H-HAQGTFTSDYSKYLDS-K (Hexadecanoyl-Iso Glu)-
AAHDFVEWLLSA-NH ₂
2 HH-NMeSer-QGTFTSDYSKYLDS-K (Hexadecanoyl-Iso Glu)-
AAHDFVEWLLSA-NH ₂
3 HH-Ac3c-QGTFTSDYSKYLDS-K (Hexadecanoyl-Iso Glu)-
AAHDFVEWLLSA-NH ₂
4 HH-Ac4c-QGTFTSDYSKYLDS-K (Hexadecanoyl-Iso Glu)-
AAHDFVEWLLSA-NH ₂
5 H-HSHGTFTSDYSKYLDS-K (Hexadecanoyl-Iso Glu)-
AAHDFVEWLLSA-NH ₂
6 H-HAHGTFTSDYSKYLDS-K (Hexadecanoyl-Iso Glu)-
AAHDFVEWLLSA-NH ₂
7 HH-DAla-HGTFTSDYSKYLDS-K (Hexadecanoyl-Iso Glu)-
AAHDFVEWLLSA-NH ₂
8 HH-Ac3c-HGTFTSDYSKYLDS-K (Hexadecanoyl-Iso Glu)-
AAHDFVEWLLSA-NH ₂
9 HH-Ac4c-HGTFTSDYSKYLDS-K (Hexadecanoyl-Iso Glu)-
AAHDFVEWLLSA-NH ₂
10 HH-Abu-QGTFTSDYSKYLDE-K (Hexadecanoyl-Iso Glu)-
AAKDFIEWLESA-NH ₂
11 H-HAQGTFTSDYSKYLDE-K (Hexadecanoyl-Iso Glu)-
AAKDFIEWLESA-NH ₂
12 HH-DAla-QGTFTSDYSKYLDE-K (Hexadecanoyl-Iso Glu)-
AAKDFIEWLESA-NH ₂
13 H-HPQGTFTSDYSKYLDE-K (Hexadecanoyl-Iso Glu)-
AAKDFIEWLESA-NH ₂
14 HH-Ac4c-QGTFTSDYSKYLDE-K (Hexadecanoyl-Iso Glu)-
AAKDFIEWLESA-NH ₂
15 HH-Ac4c-HGTFTSDYSKYLDE-K (Hexadecanoyl-Iso Glu)-
AAKDFIEWLESA-NH ₂
16 HY-Aib-QGTFTSDYSKYLDE-K (Hexadecanoyl-Iso Glu)-
AAKDFIEWLESA-NH ₂
17 HH-Aib-QGTFTSDYSKYLDE-K (Hexadecanoyl-Iso Glu)-
AAKDFIEWLEEE-NH ₂

The modification of K (hexadecanoyl-isoGlu) is as described above.

Example 2
Glucagon Receptor and GLP-1 Receptor Efficacy Assay Encodes Human Glucagon Receptor (Glucagon-R) (Primary Accession No. P47871) or Human Glucagon-like Peptide-1 Receptor (GLP-1R) (Primary Accession No. P43220) cDNA was synthesized and cloned into a mammalian expression vector containing a Zeocin resistance marker.

A mammalian expression vector encoding glucagon-R or GLP-1-R was transfected into Chinese hamster ovary (CHO) cells by the Attractene method. Zeocin selection (250 μg / ml) by limiting dilution of cells resistant to selection pressure gave stable expression clones. Cell clones expressing glucagon-R and GLP-1-R were picked up, proliferated and tested in the glucagon-R and GLP-1-R potency assay described below. One glucagon-R expression clone and one GLP-1-R expression clone were selected for compound profiling.

CHO cells expressing human glucagon-R or human GLP-1-R were seeded and assayed 24 hours later with 30,000 cells in a culture of 100 μL growth medium per well using a 96-well microtiter plate. did. On the day of analysis, growth medium was removed and cells were washed once with 200 μL assay buffer (Krebs-Ringer-buffer-KRBH). Remove buffer and incubate in 10 μL KRBH (KRBH + 10 mM HEPES, 5 mM LVDS3, 0.1% (v / v) BSA) with 0.1 mM IBM X in deionized water for 15 minutes at room temperature with each concentration of test peptide. did. The reaction was stopped by the addition of lysis buffer (0.1% w / v BSA, 5 mM HEPES, 0.3% v / v Tween20). After lysing the cells for 10 minutes at room temperature, the lysate was transferred to a 384-well plate and a 10 μL acceptor / donor bead mixture from the AlphaScreen ™ cAMP Functional Assay Kit was added. After incubation in the dark at room temperature for 1 hour, cAMP content was measured using Perkin-Elmer's AlphaScreen ™ cAMP Functional Assay Kit and according to the manufacturer's instructions. By applying a computer-aided curve fitting, the reference compound was calculated relative potency and EC ₅₀ s were compared (glucagon and GLP-1) and. The GLP-1 / glucagon ratio was calculated as described above. See Table 1.

Example 3
Agonist activity on the endogenous GLP-1 receptor The agonist activity of the test compound on the endogenous GLP-1 receptor was determined using a mouse insulinoma cell line. Intracellular cAMP was used as an indicator of receptor activation.

Cells were cultured in 384-well plates at a density of 10,000 cells / well for 24 hours. Remove the medium and add 10 μL of KRBH buffer (NaCl 130 mM, KCl 3.6 mM, NaH) containing the test compound or GLP-1 (each concentration from 0.1 pM to 100 nM) or solvent control (0.1% (v / v) DMSO). ₂ PO ₄ 0.5 mM, DDL ₄ 0.5 mM, CaCl ₂ 1.5 mM) was added to the wells at a temperature of 26 ° C. for 15 minutes.

Cellular cAMP content was measured using the AlphaScreen cAMP Functional Assay Kit (Perkin Elmer). Measurements were performed using Envision (Perkin Elmer) according to the manufacturer's recommendations.

All measurements were quadruple measurements.

Results were converted to cAMP concentrations using the cAMP standard curve prepared in KRBH buffer containing 0.1% (v / v) DMSO. The obtained cAMP curve was plotted in a log as the absolute cAMP concentration (nM) (test compound concentration) and analyzed using the curve fitting program XLfit.

The parameters calculated to explain the efficacy and agonist activity of each test compound on the endogenous GLP-1 receptor are pEC ₅₀ ( _{negative log value of EC 50} , concentration giving a semi-maximum increase in cAMP level, test compound The percentage (% CTL) relative to the control (reflecting efficacy) is (% cAMP increase for each test compound concentration standardized based on the GLP-1-induced maximal cAMP response (100% CTL)). .. See Table 2.

Example 4
Agonist activity on endogenous glucagon receptors The agonist activity of the test compound on endogenous glucagon receptors was determined by measuring their effect on the rate of glycogen synthesis in primary cultured hepatocytes. At the time of activation of glycogen receptors, inhibition of glycogen synthesis rate is predicted. The rate of glycogen synthesis was measured by measuring the amount of radioactivity-labeled glucose taken into the cellular glycogen storage over a period of time.

Primary rat hepatocytes were cultured in a 24-well plate at a density of 40,000 cells / well at 37 ° C. for 24 hours _{at 5% CO 2.}

The medium was discarded and the cells were washed with PBS. Next, 180 μL of KRBH-based buffer containing 0.1% BSA and 22.5 mM glucose was added to the wells, followed by the test compound and 40 μCi / ml D- [U14C] glucose (20 μL each). Incubation was continued for 3 hours.

At the end of the incubation period, the incubation buffer was aspirated, the cells were washed once with ice-cold PBS and then incubated with 100 μL 1 mol / l NaOH for 30 minutes at room temperature to dissolve.

Glycogen was precipitated by transferring the cell lysates to a 96-well filter plate and incubating the filter plate at 4 ° C. for 120 minutes, then the filter plate was washed 4 times with ice-cold ethanol (70%). The resulting precipitate was filtered and dried, and ^{the amount of 14} C-glucose taken up was measured using a Topcount scintillation counter according to the manufacturer's recommendations.

Wells containing vehicle control (0.1% (v / v) DMSO in KRBH buffer) were included as a reference for glycogen synthesis when uninhibited (100% CTL). Wells without D- [U ¹⁴ C] glucose were included as controls for non-specific background signals (divide this value from all values). The endogenous glucagon peptide was used as a positive control.

All treatments were performed with at least triple measurements.

The parameters calculated to account for the efficacy and agonist activity of each test compound on the endogenous glucagon receptor are pEC ₅₀ and% CTL.

Background After dividing the CPM / well, calculate as the ratio of CPM / well in the presence of the test compound to the vehicle control CPM / well to determine% CTL:
% CTL = [CPM / well (base) -CPM / well (sample)] * 100 / [CPM / well (base) -CPM / well (control)]

The glucagon receptor activator inhibits the rate of glycogen synthesis, resulting in a% CTL value between 0% CTL (complete inhibition) and 100% CTL (no observable inhibition).

The obtained activity curve was plotted in log as an absolute count (unit: cpm / sample) (test compound concentration) and analyzed using the curve fitting program XLfit.

pEC ₅₀ ( _{negative logarithmic value of EC 50} ) reflects the potency of the test compound.

_{The terms EC 50} and pEC ₅₀ used for GLP-1R activation could be _{IC 50} and pIC ₅₀ for glycogen synthesis as well.

Claims (23)

Compound with formula R ¹ -X- R ² [in the formula,
R ¹ is H (ie hydrogen), C _1-4 alkyl, acetyl, formyl, benzoyl, or trifluoroacetyl;
R ² is OH or NH ₂ ;
X is an array:
A peptide having His-X2-X3-GTFTSDYSKYL-X15-X16-X17-X18-A-X20-DFI-X24-WLE-X28-Ala;
X2 is selected from Ac3c, Ac4c, and Ac5c;
X3 is selected from Gln and His;
X15 is selected from Asp and Glu;
X16 is selected from Glu, Lys and Ψ;
X17 is selected from Lys, Arg and Ψ;
X18 is selected from Ala and Arg;
X20 is selected from Lys and Ψ;
X24 is selected from Glu, Lys and Ψ;
X28 is selected from Ser, Lys and Ψ;
Wherein said compound comprises up to 1 residue Ψ is the residue of Lys, and the side chains of the residues Ψ is attached to the lipophilic substituent]
Alternatively, a pharmaceutically acceptable salt or solvate thereof. Peptide X
H-Ac4c-QGTFTSDYSKYLDEKAAKDFIEWLESA
H-Ac4c-HGTFTSDYSKYLDEKAAKDFIEWLESA
H-Ac4c-QGTFTSDYSKYLDEKRAKDFIEWLESA
H-Ac4c-QGTFTSDYSKYLDKRAAKDFIEWLESA
H-Ac4c-QGTFTSDYSKYLDEKRAKDFIEWLESA
H-Ac4c-QGTFTSDYSKYLDERAAKDFIKWLESA
H-Ac4c-QGTFTSDYSKYLDERRAKDFIKWLESA
H-Ac4c-QGTFTSDYSKYLDERAAKDFIEWLEKA
H-Ac4c-QGTFTSDYSKYLDERRAKDFIEWLEKA and
H-Ac4c-HGTFTSDYSKYLDEKRAKDFIEWLESA
The compound according to claim 1, which has a sequence selected from. HH-Ac4c-QGTFTSDYSKYLDEKAAKDFIEWLESA-NH ₂
HH-Ac4c-HGTFTSDYSKYLDEKAAKDFIEWLESA-NH ₂
HH-Ac4c-QGTFTSDYSKYLDEKRAKDFIEWLESA-NH ₂
HH-Ac4c-QGTFTSDYSKYLDKRAAKDFIEWLESA-NH ₂
HH-Ac4c-QGTFTSDYSKYLDEKRAKDFIEWLESA-NH ₂
HH-Ac4c-QGTFTSDYSKYLDERAAKDFIKWLESA-NH ₂
HH-Ac4c-QGTFTSDYSKYLDERRAKDFIKWLESA-NH ₂
HH-Ac4c-QGTFTSDYSKYLDERAAKDFIEWLEKA-NH ₂
HH-Ac4c-QGTFTSDYSKYLDERRAKDFIEWLEKA-NH ₂ and
HH-Ac4c-HGTFTSDYSKYLDEKRAKDFIEWLESA-NH ₂
The compound according to claim 2, which is selected from. Peptide X
H-Ac4c-QGTFTSDYSKYLDEΨAAKDFIEWLESA
H-Ac4c-HGTFTSDYSKYLDEΨAAKDFIEWLESA
H-Ac4c-QGTFTSDYSKYLDEΨRAKDFIEWLESA
H-Ac4c-QGTFTSDYSKYLDΨRAAKDFIEWLESA
H-Ac4c-QGTFTSDYSKYLDEΨRAKDFIEWLESA
H-Ac4c-QGTFTSDYSKYLDERAAKDFIΨWLESA
H-Ac4c-QGTFTSDYSKYLDERRAKDFIΨWLESA
H-Ac4c-QGTFTSDYSKYLDERAAKDFIEWLEΨA
H-Ac4c-QGTFTSDYSKYLDERRAKDFIEWLEΨA and
H-Ac4c-HGTFTSDYSKYLDEΨRAKDFIEWLESA
The compound according to claim 1, which has a sequence selected from. HH-Ac4c-QGTFTSDYSKYLDEΨAAKDFIEWLESA-NH ₂
HH-Ac4c-HGTFTSDYSKYLDEΨAAKDFIEWLESA-NH ₂
HH-Ac4c-QGTFTSDYSKYLDEΨRAKDFIEWLESA-NH ₂
HH-Ac4c-QGTFTSDYSKYLDΨRAAKDFIEWLESA-NH ₂
HH-Ac4c-QGTFTSDYSKYLDEΨRAKDFIEWLESA-NH ₂
HH-Ac4c-QGTFTSDYSKYLDERAAKDFIΨWLESA-NH ₂
HH-Ac4c-QGTFTSDYSKYLDERRAKDFIΨWLESA-NH ₂
HH-Ac4c-QGTFTSDYSKYLDERAAKDFIEWLEΨA-NH ₂
HH-Ac4c-QGTFTSDYSKYLDERRAKDFIEWLEΨA-NH ₂ and
HH-Ac4c-HGTFTSDYSKYLDEΨRAKDFIEWLESA-NH ₂
The compound according to claim 4, which is selected from. The compound according to claim 5, which is HH-Ac4c-QGTFTSDYSKYLDERAAKDFIΨWLESA-NH _2. If the residue Ψ is present
The lipophilic substituent is the formula Z ¹ (in the formula, Z ¹ is the lipophilic moiety directly attached to the side chain of residue Ψ) or Z ¹ Z ² (in the formula, Z ¹ is lipophilic). Partial, Z ² is a spacer, and Z ¹ is attached to the side chain of residue Ψ via ^{Z 2).}
The compound according to any one of claims 1 to 6. The compound according to claim 7 , wherein Ψ is Lys (hexadecanoyl-isoGlu). H-Ac4c-QGTFTSDYSKYLDE-K (Hexadecanoyl-Iso Glu)-AAKDFIEWLESA or
H-Ac4c-HGTFTSDYSKYLDE-K (Hexadecanoyl-Iso Glu)-AAKDFIEWLESA
The compound according to claim 1, which has the sequence of. HH-Ac4c-QGTFTSDYSKYLDE-K (Hexadecanoyl-Iso Glu)-AAKDFIEWLESA-NH ₂ and
HH-Ac4c-HGTFTSDYSKYLDE-K (Hexadecanoyl-Iso Glu)-AAKDFIEWLESA-NH ₂
The compound according to claim 1, which is selected from. A pharmaceutically acceptable salt of the compound according to any one of claims 2 to 10. A composition comprising the compound according to any one of claims 1 to 11 or a pharmaceutically acceptable salt thereof in a mixture with a carrier. The composition according to claim 12 , wherein the composition is a pharmaceutical composition, and the carrier is a pharmaceutically acceptable carrier. The compound according to any one of claims 1 to 10, for use in medical treatment. The compound according to any one of claims 1 to 11 or a compound thereof for use in a method for preventing weight gain or promoting weight loss in an individual in which it is necessary to prevent weight gain or promote weight loss. A pharmaceutical composition comprising a pharmaceutically acceptable salt. In the required individual to increase the reduction and / or HDL / LDL ratio circulating LDL levels, for use in a method of increasing the reduction and / or HDL / LDL ratio circulating LDL levels, according to claim 1 to 11 A pharmaceutical composition containing the compound according to any one item or a pharmaceutically acceptable salt thereof. A pharmaceutical composition comprising the compound according to any one of claims 1 to 11 or a pharmaceutically acceptable salt thereof for use in the treatment of a symptom caused by or characterized by overweight. .. Obesity, morbid obesity, preoperative morbid obesity, obesity-related inflammation, obesity-related biliary sac disease, obesity-induced sleep aspiration, diabetes, metabolic syndrome, hypertension, atherosclerosis, atherosclerosis The compound according to any one of claims 1 to 11 or a pharmaceutical drug thereof for use in a method for preventing or treating obesity, arteriosclerosis, coronary heart disease, peripheral arterial disease, stroke or microvascular disease. A pharmaceutical composition comprising an acceptable salt. The pharmaceutical composition according to any one of claims 15 to 18 , for administration as part of a combination therapy with an agent for the treatment of diabetes, obesity, dyslipidemia, or hypertension. The agents for the treatment of diabetes include biguanides (eg, metformin), sulfonylureas, meglitinides or glinides (eg, nateglinide), DPP-IV inhibitors, SGLT2 inhibitors, glitazones, different GLP-1 agonists, insulin or insulin analogs. The pharmaceutical composition according to claim 19 , which is a body. The agents for the treatment of obesity include glucagon-like peptide receptor 1 agonist, peptide YY receptor agonist or its analogs, cannabinoid receptor 1 antagonist, lipase inhibitor, melanocortin receptor 4 agonist, melanin aggregation hormone receptor 1 Claim 19 is an antagonist, fentermine, a combination of fentelmin and topiramate, a combination of a norepinephrine / dopamine reuptake inhibitor and an opioid receptor antagonist (eg, a combination of bupropion and naltrexone), or a serotonin agonist. The pharmaceutical composition described. The pharmaceutical composition according to claim 19 , wherein the agent for the treatment of hypertension is an angiotensin converting enzyme inhibitor, angiotensin II receptor blocker, diuretic, beta blocker, or calcium channel blocker. 19. The pharmaceutical composition of claim 19, wherein the agent for the treatment of dyslipidemia is a statin, fibrate, niacin, and / or cholesterol absorption inhibitor.